From previous studies we have improved our understanding considerably of the mechanism by which adenylate cyclase is regulated by hormones, fluoride, guanine nucleotide, and adenosine, and the effects that these agents have on the enzyme's affinity for divalent cation at the cation binding site. We have improved the assay of adenylate cyclase by developing an enzymatic procedure for the preparation of (alpha-32P)ATP, by characterizing the influence of nucleotide pyrophosphatase on cyclase activity, and by describing means by which that influence can be minimized. We have described the dispersion by detergents of enzyme from brain and liver plasma membranes and have achieved some success in stabilizing the dispersed enzyme and in partially purifying it by ion exchange and hydrophobic chromatography. We intend to build on this experience: a) To clarify the mechanisms whereby adenylate cyclase is modulated by NAD and by oxidizing or reducing environments; b) To identify and the purify additional factors that may be involved in the regulation of adenylate cyclase; c) To establish whether there is a casual relationship between membrane phosphorylation and the control of adenylate cyclase by glucagon, fluoride, guanine nucleotides, and cholera toxin; and d) To develop and then employ several new hydrophobic and affinity chromatography systems that would find specific application in the purification of adenylate cyclase and its regulatory subunits, but also general application with other enzymes utilizing cations or nucleosides, or which are membrane bound.